Structural basis for activity of and resistance to HIV integrase inhibitors

摘要 人类免疫缺陷病毒1型(HIV-1，以下简称HIV)目前感染了约4000万人 世界各地的人，并且感染人数继续上升。在没有治愈方法的情况下，抗逆转录病毒 治疗是主要的治疗选择，因为它减缓了疾病的进展，减少了新的 感染。整合酶(IN)链转移抑制药(INSTI)是一类可阻断 病毒DNA整合到宿主染色体中，这是一个由病毒IN酶介导的过程，该酶 在病毒DNA末端组装成低聚核蛋白复合体，称为“内聚体”。INSTIS 选择性地针对肠系膜，代表临床上的一线治疗方法。然而，IN变种的出现 对INSTIs的耐药性正在成为一个更大的临床问题。结构生物学方法可以揭示 药物作用和耐药性的潜在机制，为合理改善 最新的INSTI。当辅助性技术，如生化活性分析，生物物理 热力学和动力学测量、细胞病毒学和各种计算方法，包括 自由能计算，这些结构精确地详细说明了抵抗特定INSTIs和 为设计和开发抗击感染的新型第三代INSTI提供指导。在这份提案中， 围绕利用低温电子显微镜的革命性进展进行结构研究的方法将 展示INSTI如何与其天然药物靶标HIV intasome(WT和突变体)相互作用，并阐明 对这些药物产生抗药性的机制。具体目标有三个：(1)延伸 并在当前努力的基础上，提供对为什么以及如何选择病毒抗性的机械性理解 变种(VRV)对临床使用的药物多洛替格韦(DTG)或最有效的药物产生反应 开发的内部化合物4d，目前正在进行临床前评估；(2)广泛识别 并分析了新的耐药机制和途径，出现了对第二次治疗的反应 生成药物，强调原发突变和补偿突变，并提供预测策略 未来的变种；(3)对新的第三代治疗反应产生的残留抗性变种进行选择 基于底物模仿的概念合成的INSTI，其中许多有效地抑制病毒 第二代临床使用的INSTI药物治疗后出现的耐药变异，并解释 新化合物超强效力的潜在机制。这项工作将提高我们对 一类重要的药物，用于治疗艾滋病毒携带者，识别感染艾滋病毒的机制、途径和模式 与临床相关的INSTI耐药性，并为其合理改善提供具体指南。

ABSTRACT The Human Immunodeficiency Virus Type 1 (HIV-1, hereafter referred to as HIV) currently infects ~40 million people worldwide, and the number of infected individuals continues to rise. In the absence of a cure, antiretroviral therapy represents the primary treatment option, because it slows disease progression and reduces new infections. Integrase (IN) Strand Transfer Inhibitors (INSTIs) are a class of antiretroviral therapeutics that block integration of viral DNA into host chromosomes, a process that is mediated by the viral IN enzyme, which assembles into oligomeric nucleoprotein complexes on the ends of viral DNA, termed “intasomes”. INSTIs selectively target intasomes and represent first-line therapies in the clinic. However, the emergence of IN variants resistant to INSTIs is becoming a greater clinical problem. Structural biology approaches can shed light on the mechanisms underlying drug action and resistance, providing useful information for rationally improving the current INSTIs. When complemented with ancillary techniques, such as biochemical activity assays, biophysical thermodynamic and kinetic measurements, cellular virology, and diverse computational approaches including free energy calculations, the structures precisely detail mechanisms of resistance against specific INSTIs and provide guidance for designing and developing novel 3rd generation INSTIs to fight infections. In this proposal, approaches centered around using revolutionary advances in cryo-electron microscopy for structural studies will show how INSTIs interact with their natural drug target, the HIV intasome (both WT and mutant), and elucidate the mechanisms by which resistance to these drugs emerges. There are three Specific Aims that will: (1) extend and build upon current efforts to provide a mechanistic understanding of both why and how select viral resistant variants (VRVs) arise in response to the clinically used drug Dolutegravir (DTG) or the most potent developmental in-house compound that 4d, which is currently under pre-clinical evaluation; (2) broadly identify and analyze novel mechanisms and pathways of drug resistance that arise in response to treatment with 2nd generation drugs, highlighting both primary and compensatory mutations, and providing strategies to predict future variants; (3) select for residual resistant variants arising in response to treatment with novel 3rd generation INSTIs that were synthesized based on the concept of substrate mimicry, many of which effectively inhibit viral resistant variants that arise in response to treatment with 2nd generation clinically used INSTI drugs, and explain mechanisms underlying the superior potency of novel compounds. This work will improve our understanding of an important class of drugs used to treat people living with HIV, identify mechanisms, pathways, and patterns of clinically relevant resistance to INSTIs, and provide specific guidelines for their rational improvement.